DESCRIPTION: Thiol groups play significant roles in various normal and abnormal cellular functions. Thiol status is compartmentalized at the subcellular level with the most reducing to the most oxidizing in the order of mitochondria > nuclei > cytoplasm > endoplasmic reticulum > extracellular space. Thiol status is affected by various physiological, pathological and environmental factors. A change in thiol status in mitochondria/nucleus has been associated with aging and various disease states such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). Current understanding of the impacts of thiol changes on these diseases is mostly based on research through thiol concentration determination using isolated mitochondria/nuclei. Many details and specifics remain unknown on thiols' role in the pathophysiology of these diseases. This application is aimed to develop thiol specific fluorogenic agents that can be used to image and quantify thiols in mitochondria/nuclei in real-time in live cells through fluorescence microscopy such as confocal microscopy and multi- photon (mostly two-photon) microscopy. These agents can provide detailed, specific, dynamic, and potentially molecular level information on thiols in mitochondria/nuclei in its intact and native physiological environment compared with most other analytical methods that provide information only on thiol concentration. The specific information can help us better understand thiols' role in the pathophysiology of the diseases and help prevent and find better treatment of the diseases. Currently, there is no agent that can be used to image and quantify thiols in mitochondria/nuclei in live cells through fluorescence microscopy. Our proposed work is based on our recent finding of a novel thiol specific sulfide-thiol exchange reaction. Based on this reaction, we have developed a class of benzofurazan sulfides that convert thiols specifically, rapidly, and completely to fluorescent molecules. One of the benzofurazan sulfides was selected to demonstrate the ability to image and quantify thiols in live cells through fluorescence microscopy. The work was reported recently in Analytical Chemistry. In this proposed work, we will take advantage of this novel finding and extend it to the development of agents that can image and quantify thiols in mitochondria/nuclei. We will link a benzofurazan sulfide with a well-established mitochondria or nucleus targeting molecule. These designed molecules will be synthesized. Their one-photon and two- photon fluorescence properties will be determined, and their ability to image and quantify thiols in mitochondria and nuclei will be evaluated in three different cell lines. The long term goal of this project is to develop agents that can image and quantify thiols in other subcellular organelles by linking a benzofurazan sulfide to a subcellular targeting molecule. These agents will be valuable tools in investigating thiols' roles in various disease states at the subcellular levels.